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Joseph Heitman - One of the best experts on this subject based on the ideXlab platform.
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a novel resistance pathway for calcineurin inhibitors in the human pathogenic Mucorales Mucor circinelloides
Mbio, 2020Co-Authors: Sandeep Vellanki, Joseph Heitman, Blake R Billmyre, Eun Young Huh, Alejandra Lorenzen, Micaela Campbell, Broderick Turner, Soo Chan LeeAbstract:ABSTRACT Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of Mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi, and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed 36 calcineurin inhibitor-resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRΔ mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin). bycA encodes an amino acid permease. We verified that both the bycAΔ single mutant and the bycAΔ cnbRΔ double mutant are resistant to calcineurin inhibitor FK506, thereby demonstrating a novel mechanism of resistance against calcineurin inhibitors. We also found that the level of expression of bycA was significantly higher in the wild-type strain treated with FK506 and in the cnbRΔ mutants but was significantly lower in the wild-type strain without FK506 treatment. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and that calcineurin suppresses expression of the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hypha-yeast transition as our data demonstrate positive correlations among bycA expression, protein kinase A activity, and Mucor yeast growth. Also, calcineurin, independently of its role in morphogenesis, contributes to virulence traits, including phagosome maturation blockade, host cell damages, and proangiogenic growth factor induction during interactions with hosts. IMPORTANCEMucor is intrinsically resistant to most known antifungals, which makes Mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase that is widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, growth shifts to a less virulent yeast growth form, which makes calcineurin an attractive target for development of new antifungal drugs. Previously, we identified two distinct mechanisms through which Mucor can become resistant to calcineurin inhibitors involving Mendelian mutations in the gene for FKBP12, including mechanisms corresponding to calcineurin A or B subunits and epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism where loss-of-function mutations in the amino acid permease corresponding to the bycA gene contribute to resistance against calcineurin inhibitors. When calcineurin activity is absent, BycA can activate protein kinase A (PKA) to promote yeast growth via a cAMP-independent pathway. Our data also show that calcineurin activity contributes to host-pathogen interactions primarily in the pathogenesis of Mucor.
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A Novel Resistance Pathway for Calcineurin Inhibitors in the Human-Pathogenic Mucorales Mucor circinelloides
'American Society for Microbiology', 2020Co-Authors: Sandeep Vellanki, Joseph Heitman, Blake R Billmyre, Eun Young Huh, Alejandra Lorenzen, Micaela Campbell, Broderick Turner, Soo Chan LeeAbstract:Mucor is intrinsically resistant to most known antifungals, which makes Mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase that is widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, growth shifts to a less virulent yeast growth form, which makes calcineurin an attractive target for development of new antifungal drugs. Previously, we identified two distinct mechanisms through which Mucor can become resistant to calcineurin inhibitors involving Mendelian mutations in the gene for FKBP12, including mechanisms corresponding to calcineurin A or B subunits and epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism where loss-of-function mutations in the amino acid permease corresponding to the bycA gene contribute to resistance against calcineurin inhibitors. When calcineurin activity is absent, BycA can activate protein kinase A (PKA) to promote yeast growth via a cAMP-independent pathway. Our data also show that calcineurin activity contributes to host-pathogen interactions primarily in the pathogenesis of Mucor.Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of Mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi, and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed 36 calcineurin inhibitor-resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRΔ mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin). bycA encodes an amino acid permease. We verified that both the bycAΔ single mutant and the bycAΔ cnbRΔ double mutant are resistant to calcineurin inhibitor FK506, thereby demonstrating a novel mechanism of resistance against calcineurin inhibitors. We also found that the level of expression of bycA was significantly higher in the wild-type strain treated with FK506 and in the cnbRΔ mutants but was significantly lower in the wild-type strain without FK506 treatment. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and that calcineurin suppresses expression of the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hypha-yeast transition as our data demonstrate positive correlations among bycA expression, protein kinase A activity, and Mucor yeast growth. Also, calcineurin, independently of its role in morphogenesis, contributes to virulence traits, including phagosome maturation blockade, host cell damages, and proangiogenic growth factor induction during interactions with hosts
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a novel resistance pathway for calcineurin inhibitors in the human pathogenic Mucorales Mucor circinelloides
bioRxiv, 2019Co-Authors: Sandeep Vellanki, Joseph Heitman, Blake R Billmyre, Eun Young Huh, Alejandra Lorenzen, Micaela Campbell, Broderick Turner, Soo Chan LeeAbstract:Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of Mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRΔ mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin A). bycA encodes an amino acid permease. We verified that both bycAΔ, and the bycAΔ cnbRΔ double mutant are resistant to the calcineurin inhibitor FK506, thereby demonstrating a novel resistance mechanism against calcineurin inhibitors. We also found that the expression of bycA was significantly higher in the wild type strain treated with FK506 and in the cnbRΔ mutants, but significantly lower in the wild type without FK506. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and calcineurin suppresses the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hyphal-yeast transition as our data demonstrates a positive correlation between bycA expression, protein kinase A activity, and Mucor yeast-growth. Also calcineurin, independent of its role in morphogenesis, contributes to virulence traits including phagosome maturation blockade, host cell damages, and pro-angiogenic growth factor induction during interactions with hosts.
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a novel resistance pathway for calcineurin inhibitors in the human pathogenic Mucorales Mucor circinelloides
bioRxiv, 2019Co-Authors: Sandeep Vellanki, Joseph Heitman, Blake R Billmyre, Eun Young Huh, Alejandra Lorenzen, Micaela Campbell, Broderick Turner, Soo Chan LeeAbstract:Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides (Mucor)is a causal agent of Mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conservedvirulence factor in many pathogenic fungi and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results ina shift from hyphal to yeast growth. We analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRDmutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named herebycA(bypass of calcineurinA). bycAencodes an amino acid permease. We verified that both bycAD, and the bycADcnbRDdouble mutantare resistant tothe calcineurin inhibitor FK506, thereby demonstrating a novel resistance mechanism against calcineurin inhibitors. We also found that the expression of bycA was significantly higher in the wild type with FK506 and in the cnbRDmutants, but significantly lower in the wild type without FK506. These findings suggest that bycAis a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucorand calcineurin suppresses thebycA geneat the mRNA levelto promote hyphal growth. BycA is involved in the Mucorhyphal-yeast transition as our data demonstrates a positive correlation betweenbycA expression, protein kinase A activity, and Mucor yeast-growth. Also, calcineurin activity rather than hyphal morphology primarily contributes to virulence traits including phagosome maturation blockade, host cell damages, and pro-angiogenic growth factor induction during interactions with hosts.
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drug resistant epimutants exhibit organ specific stability and induction during murine infections caused by the human fungal pathogen Mucor circinelloides
Mbio, 2019Co-Authors: Zanetta Chang, Joseph HeitmanAbstract:ABSTRACT The environmentally ubiquitous fungus Mucor circinelloides is a primary cause of the emerging disease Mucormycosis. Mucor infection is notable for causing high morbidity and mortality, especially in immunosuppressed patients, while being inherently resistant to the majority of clinically available antifungal drugs. A new, RNA interference (RNAi)-dependent, and reversible epigenetic mechanism of antifungal resistance—epimutation—was recently discovered in M. circinelloides. However, the effects of epimutation in a host-pathogen setting were unknown. We employed a systemic, intravenous murine model of Mucor infection to elucidate the potential impact of epimutation in vivo. Infection with an epimutant strain resistant to the antifungal agents FK506 and rapamycin revealed that the epimutant-induced drug resistance was stable in vivo in a variety of different organs and tissues. Reversion of the epimutant-induced drug resistance was observed to be more rapid in isolates from the brain than in isolates recovered from the liver, spleen, kidney, or lungs. Importantly, infection with a wild-type strain of Mucor led to increased rates of epimutation after strains were recovered from organs and exposed to FK506 stress in vitro. Once again, this effect was more pronounced in strains recovered from the brain than from other organs. In summary, we report the rapid induction and reversion of RNAi-dependent drug resistance after in vivo passage through a murine model, with pronounced impact in strains recovered from brain. Defining the role played by epimutation in drug resistance and infection advances our understanding of Mucor and other fungal pathogens and may have implications for antifungal therapy. IMPORTANCE The emerging fungal pathogen Mucor circinelloides causes a severe infection, Mucormycosis, which leads to considerable morbidity and mortality. Treatment of Mucor infection is challenging because Mucor is inherently resistant to nearly all clinical antifungal agents. An RNAi-dependent and reversible mechanism of antifungal resistance, epimutation, was recently reported for Mucor. Epimutation has not been studied in vivo, and it was unclear whether it would contribute to antifungal resistance observed clinically. We demonstrate that epimutation can both be induced and reverted after in vivo passage through a mouse; rates of both induction and reversion are higher after brain infection than after infection of other organs (liver, spleen, kidneys, or lungs). Elucidating the roles played by epimutation in drug resistance and infection will improve our understanding of Mucor and other fungal pathogens and may have implications for antifungal treatment.
Soo Chan Lee - One of the best experts on this subject based on the ideXlab platform.
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a novel resistance pathway for calcineurin inhibitors in the human pathogenic Mucorales Mucor circinelloides
Mbio, 2020Co-Authors: Sandeep Vellanki, Joseph Heitman, Blake R Billmyre, Eun Young Huh, Alejandra Lorenzen, Micaela Campbell, Broderick Turner, Soo Chan LeeAbstract:ABSTRACT Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of Mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi, and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed 36 calcineurin inhibitor-resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRΔ mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin). bycA encodes an amino acid permease. We verified that both the bycAΔ single mutant and the bycAΔ cnbRΔ double mutant are resistant to calcineurin inhibitor FK506, thereby demonstrating a novel mechanism of resistance against calcineurin inhibitors. We also found that the level of expression of bycA was significantly higher in the wild-type strain treated with FK506 and in the cnbRΔ mutants but was significantly lower in the wild-type strain without FK506 treatment. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and that calcineurin suppresses expression of the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hypha-yeast transition as our data demonstrate positive correlations among bycA expression, protein kinase A activity, and Mucor yeast growth. Also, calcineurin, independently of its role in morphogenesis, contributes to virulence traits, including phagosome maturation blockade, host cell damages, and proangiogenic growth factor induction during interactions with hosts. IMPORTANCEMucor is intrinsically resistant to most known antifungals, which makes Mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase that is widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, growth shifts to a less virulent yeast growth form, which makes calcineurin an attractive target for development of new antifungal drugs. Previously, we identified two distinct mechanisms through which Mucor can become resistant to calcineurin inhibitors involving Mendelian mutations in the gene for FKBP12, including mechanisms corresponding to calcineurin A or B subunits and epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism where loss-of-function mutations in the amino acid permease corresponding to the bycA gene contribute to resistance against calcineurin inhibitors. When calcineurin activity is absent, BycA can activate protein kinase A (PKA) to promote yeast growth via a cAMP-independent pathway. Our data also show that calcineurin activity contributes to host-pathogen interactions primarily in the pathogenesis of Mucor.
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A Novel Resistance Pathway for Calcineurin Inhibitors in the Human-Pathogenic Mucorales Mucor circinelloides
'American Society for Microbiology', 2020Co-Authors: Sandeep Vellanki, Joseph Heitman, Blake R Billmyre, Eun Young Huh, Alejandra Lorenzen, Micaela Campbell, Broderick Turner, Soo Chan LeeAbstract:Mucor is intrinsically resistant to most known antifungals, which makes Mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase that is widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, growth shifts to a less virulent yeast growth form, which makes calcineurin an attractive target for development of new antifungal drugs. Previously, we identified two distinct mechanisms through which Mucor can become resistant to calcineurin inhibitors involving Mendelian mutations in the gene for FKBP12, including mechanisms corresponding to calcineurin A or B subunits and epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism where loss-of-function mutations in the amino acid permease corresponding to the bycA gene contribute to resistance against calcineurin inhibitors. When calcineurin activity is absent, BycA can activate protein kinase A (PKA) to promote yeast growth via a cAMP-independent pathway. Our data also show that calcineurin activity contributes to host-pathogen interactions primarily in the pathogenesis of Mucor.Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of Mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi, and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed 36 calcineurin inhibitor-resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRΔ mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin). bycA encodes an amino acid permease. We verified that both the bycAΔ single mutant and the bycAΔ cnbRΔ double mutant are resistant to calcineurin inhibitor FK506, thereby demonstrating a novel mechanism of resistance against calcineurin inhibitors. We also found that the level of expression of bycA was significantly higher in the wild-type strain treated with FK506 and in the cnbRΔ mutants but was significantly lower in the wild-type strain without FK506 treatment. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and that calcineurin suppresses expression of the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hypha-yeast transition as our data demonstrate positive correlations among bycA expression, protein kinase A activity, and Mucor yeast growth. Also, calcineurin, independently of its role in morphogenesis, contributes to virulence traits, including phagosome maturation blockade, host cell damages, and proangiogenic growth factor induction during interactions with hosts
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a novel resistance pathway for calcineurin inhibitors in the human pathogenic Mucorales Mucor circinelloides
bioRxiv, 2019Co-Authors: Sandeep Vellanki, Joseph Heitman, Blake R Billmyre, Eun Young Huh, Alejandra Lorenzen, Micaela Campbell, Broderick Turner, Soo Chan LeeAbstract:Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of Mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRΔ mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin A). bycA encodes an amino acid permease. We verified that both bycAΔ, and the bycAΔ cnbRΔ double mutant are resistant to the calcineurin inhibitor FK506, thereby demonstrating a novel resistance mechanism against calcineurin inhibitors. We also found that the expression of bycA was significantly higher in the wild type strain treated with FK506 and in the cnbRΔ mutants, but significantly lower in the wild type without FK506. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and calcineurin suppresses the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hyphal-yeast transition as our data demonstrates a positive correlation between bycA expression, protein kinase A activity, and Mucor yeast-growth. Also calcineurin, independent of its role in morphogenesis, contributes to virulence traits including phagosome maturation blockade, host cell damages, and pro-angiogenic growth factor induction during interactions with hosts.
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a novel resistance pathway for calcineurin inhibitors in the human pathogenic Mucorales Mucor circinelloides
bioRxiv, 2019Co-Authors: Sandeep Vellanki, Joseph Heitman, Blake R Billmyre, Eun Young Huh, Alejandra Lorenzen, Micaela Campbell, Broderick Turner, Soo Chan LeeAbstract:Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides (Mucor)is a causal agent of Mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conservedvirulence factor in many pathogenic fungi and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results ina shift from hyphal to yeast growth. We analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRDmutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named herebycA(bypass of calcineurinA). bycAencodes an amino acid permease. We verified that both bycAD, and the bycADcnbRDdouble mutantare resistant tothe calcineurin inhibitor FK506, thereby demonstrating a novel resistance mechanism against calcineurin inhibitors. We also found that the expression of bycA was significantly higher in the wild type with FK506 and in the cnbRDmutants, but significantly lower in the wild type without FK506. These findings suggest that bycAis a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucorand calcineurin suppresses thebycA geneat the mRNA levelto promote hyphal growth. BycA is involved in the Mucorhyphal-yeast transition as our data demonstrates a positive correlation betweenbycA expression, protein kinase A activity, and Mucor yeast-growth. Also, calcineurin activity rather than hyphal morphology primarily contributes to virulence traits including phagosome maturation blockade, host cell damages, and pro-angiogenic growth factor induction during interactions with hosts.
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gastrointestinal microbiota alteration induced by Mucor circinelloides in a murine model
Journal of Microbiology, 2019Co-Authors: Katherine D Mueller, Joseph Heitman, Hao Zhang, Christian R Serrano, Blake R Billmyre, Eun Young Huh, Philipp Wiemann, Nancy P Keller, Yufeng Wang, Soo Chan LeeAbstract:Mucor circinelloides is a pathogenic fungus and etiologic agent of Mucormycosis. In 2013, cases of gastrointestinal illness after yogurt consumption were reported to the US FDA, and the producer found that its products were contaminated with Mucor. A previous study found that the Mucor strain isolated from an open contaminated yogurt exhibited virulence in a murine systemic infection model and showed that this strain is capable of surviving passage through the gastrointestinal tract of mice. In this study, we isolated another Mucor strain from an unopened yogurt that is closely related but distinct from the first Mucor strain and subsequently examined if Mucor alters the gut microbiota in a murine host model. DNA extracted from a ten-day course of stool samples was used to analyze the microbiota in the gastrointestinal tracts of mice exposed via ingestion of Mucor spores. The bacterial 16S rRNA gene and fungal ITS1 sequences obtained were used to identify taxa of each kingdom. Linear regressions revealed that there are changes in bacterial and fungal abundance in the gastrointestinal tracts of mice which ingested Mucor. Furthermore, we found an increased abundance of the bacterial genus Bacteroides and a decreased abundance of the bacteria Akkermansia muciniphila in the gastrointestinal tracts of exposed mice. Measurements of abundances show shifts in relative levels of multiple bacterial and fungal taxa between mouse groups. These findings suggest that exposure of the gastrointestinal tract to Mucor can alter the microbiota and, more importantly, illustrate an interaction between the intestinal mycobiota and bacteriota. In addition, Mucor was able to induce increased permeability in epithelial cell monolayers in vitro, which might be indicative of unstable intestinal barriers. Understanding how the gut microbiota is shaped is important to understand the basis of potential methods of treatment for gastrointestinal illness. How the gut microbiota changes in response to exposure, even by pathogens not considered to be causative agents of food-borne illness, may be important to how commercial food producers prevent and respond to contamination of products aimed at the public. This study provides evidence that the fungal microbiota, though understudied, may play an important role in diseases of the human gut.
T L Blundell - One of the best experts on this subject based on the ideXlab platform.
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x ray analyses of aspartic proteinases v structure and refinement at 2 0 a resolution of the aspartic proteinase from Mucor pusillus
Journal of Molecular Biology, 1993Co-Authors: M Newman, Mohammed O Badasso, Anne Cleasby, S P Wood, F Watson, P Roychowdhury, H B Jones, I J Tickle, T L BlundellAbstract:Abstract The structure of Mucor pusillus pepsin (EC 3.4.23.6), the aspartic proteinase from Mucor pusillus , has been refined to a crystallographic R -factor of 16·2% at 2·0 A resolution. The positions of 2638 protein atoms, 221 solvent atoms and a sulphate ion have been determined with an estimated root-mean-square (r.m.s.) error of 0·15 to 0·20 A. In the final model, the r.m.s. deviation from ideality for bond distances is 0·022 A, and for angle distances it is 0·050 A. Comparison of the overall three-dimensional structure with other aspartic proteinases shows that Mucor pusillus pepsin is as distant from the other fungal enzymes as it is from those of mammalian origin. Analysis of a rigid body shift of residues 190 to 302 shows that Mucor pusillus pepsin displays one of the largest shifts relative to other aspartic proteinases (14·4° relative to endothiapepsin) and that changes have occurred at the interface between the two rigid bodies to accommodate this large shift. A new sequence alignment has been obtained on the basis of the three-dimensional structure, enabling the positions of large insertions to be identified. Analysis of secondary structure shows the β-sheet to be well conserved whereas α-helical elements are more variable. A new α-helix h N 4 is formed by a six-residue insertion between positions 131 and 132. Most insertions occur in loop regions: -5 to 1 (five residues relative to porcine pepsin); 115 to 116 (six residues); 186 to 187 (four residues); 263 to 264 (seven residues); 278 to 279 (four residues); and 326 to 332 (six residues). The active site residues are highly conserved in Mucor pusillus pepsin; r.m.s. difference with rhizopuspepsin is 0·37 A for 25 C α atom pairs. However, residue 303, which is generally conserved as an aspartate, is changed to an asparagine in Mucor pusillus pepsin, possibly influencing pH optimum. Substantial changes have occurred in the substrate binding cleft in the region of S 1 and S 3 due to the insertion between 115 and 116 and the rearrangement of loop 9-13. Residue Asn219 necessitates a shift in position of substrate main-chain atoms to maintain hydrogen bonding pattern. Invariant residues Asp11 and Tyr14 have undergone a major change in conformation apparently due to localized changes in molecular structure. Both these residues have been implicated in zymogen stability and activation.
Grit Walther - One of the best experts on this subject based on the ideXlab platform.
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a new species concept for the clinically relevant Mucor circinelloides complex
Persoonia, 2020Co-Authors: Lysett Wagner, Kerstin Voigt, Ana Alastrueyizquierdo, G S De Hoog, J B Stielow, K Bensch, V U Schwartze, Oliver Kurzai, Grit WaltherAbstract:Mucor species are common soil fungi but also known as agents of human infections (Mucormycosis) and used in food production and biotechnology. Mucor circinelloides is the Mucor species that is most frequently isolated from clinical sources. The taxonomy of Mucor circinelloides and its close relatives (Mucor circinelloides complex - MCC) is still based on morphology and mating behaviour. The aim of the present study was a revised taxonomy of the MCC using a polyphasic approach. Using a set of 100 strains molecular phylogenetic analysis of five markers (ITS, rpb1, tsr1, mcm7, and cfs, introduced here) were performed, combined with phenotypic studies, mating tests and the determination of the maximum growth temperatures. The multi-locus analyses revealed 16 phylogenetic species of which 14 showed distinct phenotypical traits and were recognised as discrete species. Five of these species are introduced as novel taxa: M. amethystinus sp. nov., M. atramentarius sp. nov., M. variicolumellatus sp. nov., M. pseudocircinelloides sp. nov., and M. pseudolusitanicus sp. nov. The former formae of M. circinelloides represent one or two separate species. In the MCC, the simple presence of well-shaped zygospores only indicates a close relation of both strains, but not necessarily conspecificity. Seven species of the MCC have been implemented in human infection: M. circinelloides, M. griseocyanus, M. janssenii, M. lusitanicus, M. ramosissimus, M. variicolumellatus, and M. velutinosus.
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dna barcoding in Mucorales an inventory of biodiversity
Persoonia, 2013Co-Authors: Grit Walther, Julia Pawlowska, Ana Alastrueyizquierdo, Marta Wrzosek, J L Rodrigueztudela, Somayeh Dolatabadi, Arunaloke Chakrabarti, G S De HoogAbstract:The order Mucorales comprises predominantly fast-growing saprotrophic fungi, some of which are used for the fermentation of foodstuffs but it also includes species known to cause infections in patients with severe immune or metabolic impairments. To inventory biodiversity in Mucorales ITS barcodes of 668 strains in 203 taxa were generated covering more than two thirds of the recognised species. Using the ITS sequences, Molecular Operational Taxonomic Units were defined by a similarity threshold of 99 %. An LSU sequence was generated for each unit as well. Analysis of the LSU sequences revealed that conventional phenotypic classifications of the Mucoraceae are highly artificial. The LSU- and ITS-based trees suggest that characters, such as rhizoids and sporangiola, traditionally used in Mucoralean taxonomy are plesiomorphic traits. The ITS region turned out to be an appropriate barcoding marker in Mucorales. It could be sequenced directly in 82 % of the strains and its variability was sufficient to resolve most of the morphospecies. Molecular identification turned out to be problematic only for the species complexes of Mucor circinelloides, M. flavus, M. piriformis and Zygorhynchus moelleri. As many as 12 possibly undescribed species were detected. Intraspecific variability differed widely among Mucorealean species ranging from 0 % in Backusella circina to 13.3 % in Cunninghamella echinulata. A high proportion of clinical strains was included for molecular identification. Clinical isolates of Cunninghamella elegans were identified molecularly for the first time. As a result of the phylogenetic analyses several taxonomic and nomenclatural changes became necessary. The genus Backusella was emended to include all species with transitorily recurved sporangiophores. Since this matched molecular data all Mucor species possessing this character were transferred to Backusella. The genus Zygorhynchus was shown to be polyphyletic based on ITS and LSU data. Consequently, Zygorhynchus was abandoned and all species were reclassified in Mucor. Our phylogenetic analyses showed, furthermore, that all non-thermophilic RhizoMucor species belong to Mucor. Accordingly, RhizoMucor endophyticus was transferred to Mucor and RhizoMucor chlamydosporus was synonymised with Mucor indicus. Lecto-, epi- or neotypes were designated for several taxa.
Blake R Billmyre - One of the best experts on this subject based on the ideXlab platform.
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a novel resistance pathway for calcineurin inhibitors in the human pathogenic Mucorales Mucor circinelloides
Mbio, 2020Co-Authors: Sandeep Vellanki, Joseph Heitman, Blake R Billmyre, Eun Young Huh, Alejandra Lorenzen, Micaela Campbell, Broderick Turner, Soo Chan LeeAbstract:ABSTRACT Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of Mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi, and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed 36 calcineurin inhibitor-resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRΔ mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin). bycA encodes an amino acid permease. We verified that both the bycAΔ single mutant and the bycAΔ cnbRΔ double mutant are resistant to calcineurin inhibitor FK506, thereby demonstrating a novel mechanism of resistance against calcineurin inhibitors. We also found that the level of expression of bycA was significantly higher in the wild-type strain treated with FK506 and in the cnbRΔ mutants but was significantly lower in the wild-type strain without FK506 treatment. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and that calcineurin suppresses expression of the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hypha-yeast transition as our data demonstrate positive correlations among bycA expression, protein kinase A activity, and Mucor yeast growth. Also, calcineurin, independently of its role in morphogenesis, contributes to virulence traits, including phagosome maturation blockade, host cell damages, and proangiogenic growth factor induction during interactions with hosts. IMPORTANCEMucor is intrinsically resistant to most known antifungals, which makes Mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase that is widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, growth shifts to a less virulent yeast growth form, which makes calcineurin an attractive target for development of new antifungal drugs. Previously, we identified two distinct mechanisms through which Mucor can become resistant to calcineurin inhibitors involving Mendelian mutations in the gene for FKBP12, including mechanisms corresponding to calcineurin A or B subunits and epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism where loss-of-function mutations in the amino acid permease corresponding to the bycA gene contribute to resistance against calcineurin inhibitors. When calcineurin activity is absent, BycA can activate protein kinase A (PKA) to promote yeast growth via a cAMP-independent pathway. Our data also show that calcineurin activity contributes to host-pathogen interactions primarily in the pathogenesis of Mucor.
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A Novel Resistance Pathway for Calcineurin Inhibitors in the Human-Pathogenic Mucorales Mucor circinelloides
'American Society for Microbiology', 2020Co-Authors: Sandeep Vellanki, Joseph Heitman, Blake R Billmyre, Eun Young Huh, Alejandra Lorenzen, Micaela Campbell, Broderick Turner, Soo Chan LeeAbstract:Mucor is intrinsically resistant to most known antifungals, which makes Mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase that is widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, growth shifts to a less virulent yeast growth form, which makes calcineurin an attractive target for development of new antifungal drugs. Previously, we identified two distinct mechanisms through which Mucor can become resistant to calcineurin inhibitors involving Mendelian mutations in the gene for FKBP12, including mechanisms corresponding to calcineurin A or B subunits and epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism where loss-of-function mutations in the amino acid permease corresponding to the bycA gene contribute to resistance against calcineurin inhibitors. When calcineurin activity is absent, BycA can activate protein kinase A (PKA) to promote yeast growth via a cAMP-independent pathway. Our data also show that calcineurin activity contributes to host-pathogen interactions primarily in the pathogenesis of Mucor.Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of Mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi, and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed 36 calcineurin inhibitor-resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRΔ mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin). bycA encodes an amino acid permease. We verified that both the bycAΔ single mutant and the bycAΔ cnbRΔ double mutant are resistant to calcineurin inhibitor FK506, thereby demonstrating a novel mechanism of resistance against calcineurin inhibitors. We also found that the level of expression of bycA was significantly higher in the wild-type strain treated with FK506 and in the cnbRΔ mutants but was significantly lower in the wild-type strain without FK506 treatment. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and that calcineurin suppresses expression of the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hypha-yeast transition as our data demonstrate positive correlations among bycA expression, protein kinase A activity, and Mucor yeast growth. Also, calcineurin, independently of its role in morphogenesis, contributes to virulence traits, including phagosome maturation blockade, host cell damages, and proangiogenic growth factor induction during interactions with hosts
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a novel resistance pathway for calcineurin inhibitors in the human pathogenic Mucorales Mucor circinelloides
bioRxiv, 2019Co-Authors: Sandeep Vellanki, Joseph Heitman, Blake R Billmyre, Eun Young Huh, Alejandra Lorenzen, Micaela Campbell, Broderick Turner, Soo Chan LeeAbstract:Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of Mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRΔ mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin A). bycA encodes an amino acid permease. We verified that both bycAΔ, and the bycAΔ cnbRΔ double mutant are resistant to the calcineurin inhibitor FK506, thereby demonstrating a novel resistance mechanism against calcineurin inhibitors. We also found that the expression of bycA was significantly higher in the wild type strain treated with FK506 and in the cnbRΔ mutants, but significantly lower in the wild type without FK506. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and calcineurin suppresses the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hyphal-yeast transition as our data demonstrates a positive correlation between bycA expression, protein kinase A activity, and Mucor yeast-growth. Also calcineurin, independent of its role in morphogenesis, contributes to virulence traits including phagosome maturation blockade, host cell damages, and pro-angiogenic growth factor induction during interactions with hosts.
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a novel resistance pathway for calcineurin inhibitors in the human pathogenic Mucorales Mucor circinelloides
bioRxiv, 2019Co-Authors: Sandeep Vellanki, Joseph Heitman, Blake R Billmyre, Eun Young Huh, Alejandra Lorenzen, Micaela Campbell, Broderick Turner, Soo Chan LeeAbstract:Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides (Mucor)is a causal agent of Mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conservedvirulence factor in many pathogenic fungi and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results ina shift from hyphal to yeast growth. We analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRDmutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named herebycA(bypass of calcineurinA). bycAencodes an amino acid permease. We verified that both bycAD, and the bycADcnbRDdouble mutantare resistant tothe calcineurin inhibitor FK506, thereby demonstrating a novel resistance mechanism against calcineurin inhibitors. We also found that the expression of bycA was significantly higher in the wild type with FK506 and in the cnbRDmutants, but significantly lower in the wild type without FK506. These findings suggest that bycAis a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucorand calcineurin suppresses thebycA geneat the mRNA levelto promote hyphal growth. BycA is involved in the Mucorhyphal-yeast transition as our data demonstrates a positive correlation betweenbycA expression, protein kinase A activity, and Mucor yeast-growth. Also, calcineurin activity rather than hyphal morphology primarily contributes to virulence traits including phagosome maturation blockade, host cell damages, and pro-angiogenic growth factor induction during interactions with hosts.
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gastrointestinal microbiota alteration induced by Mucor circinelloides in a murine model
Journal of Microbiology, 2019Co-Authors: Katherine D Mueller, Joseph Heitman, Hao Zhang, Christian R Serrano, Blake R Billmyre, Eun Young Huh, Philipp Wiemann, Nancy P Keller, Yufeng Wang, Soo Chan LeeAbstract:Mucor circinelloides is a pathogenic fungus and etiologic agent of Mucormycosis. In 2013, cases of gastrointestinal illness after yogurt consumption were reported to the US FDA, and the producer found that its products were contaminated with Mucor. A previous study found that the Mucor strain isolated from an open contaminated yogurt exhibited virulence in a murine systemic infection model and showed that this strain is capable of surviving passage through the gastrointestinal tract of mice. In this study, we isolated another Mucor strain from an unopened yogurt that is closely related but distinct from the first Mucor strain and subsequently examined if Mucor alters the gut microbiota in a murine host model. DNA extracted from a ten-day course of stool samples was used to analyze the microbiota in the gastrointestinal tracts of mice exposed via ingestion of Mucor spores. The bacterial 16S rRNA gene and fungal ITS1 sequences obtained were used to identify taxa of each kingdom. Linear regressions revealed that there are changes in bacterial and fungal abundance in the gastrointestinal tracts of mice which ingested Mucor. Furthermore, we found an increased abundance of the bacterial genus Bacteroides and a decreased abundance of the bacteria Akkermansia muciniphila in the gastrointestinal tracts of exposed mice. Measurements of abundances show shifts in relative levels of multiple bacterial and fungal taxa between mouse groups. These findings suggest that exposure of the gastrointestinal tract to Mucor can alter the microbiota and, more importantly, illustrate an interaction between the intestinal mycobiota and bacteriota. In addition, Mucor was able to induce increased permeability in epithelial cell monolayers in vitro, which might be indicative of unstable intestinal barriers. Understanding how the gut microbiota is shaped is important to understand the basis of potential methods of treatment for gastrointestinal illness. How the gut microbiota changes in response to exposure, even by pathogens not considered to be causative agents of food-borne illness, may be important to how commercial food producers prevent and respond to contamination of products aimed at the public. This study provides evidence that the fungal microbiota, though understudied, may play an important role in diseases of the human gut.
